Systems, Devices, and Methods for Improving Accuracy of Biosensors Using Fill Time

ABSTRACT

Methods for determining a concentration of an analyte in a sample, and the devices and systems used in conjunction with the same, are provided herein. In one exemplary embodiment of a method for determining a concentration of an analyte in a sample, the method includes detecting a presence of a sample in an electrochemical sensor including two electrodes. A fill time of the sample is determined with the two electrodes and a correction factor is calculated in view of at least the fill time. The method also includes reacting an analyte that causes a physical transformation of the analyte between the two electrodes. A concentration of the analyte can then be determined in view of the correction factor with the same two electrodes. Systems and devices that take advantage of the fill time to make analyte concentration determinations are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 as acontinuation-in-part application to U.S. patent application Ser. No.12/649,594, entitled “Systems, Devices and Methods for ImprovingAccuracy of Biosensors Using Fill Time” filed on Dec. 30, 2009, which ishereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to determining a concentration of ananalyte in a sample, and more particularly relates to making a moreaccurate determination of the concentration based on the fill time ofthe sample.

BACKGROUND

Analyte detection in physiological fluids, e.g. blood or blood derivedproducts, is of ever increasing importance to today's society. Analytedetection assays find use in a variety of applications, includingclinical laboratory testing, home testing, etc., where the results ofsuch testing play a prominent role in diagnosis and management in avariety of disease conditions. Analytes of interest include glucose fordiabetes management, cholesterol, and the like. In response to thisgrowing importance of analyte detection, a variety of analyte detectionprotocols and devices for both clinical and home use have beendeveloped. Some of these devices include electrochemical cells,electrochemical sensors, hemoglobin sensors, antioxidant sensors,biosensors, and immunosensors.

One characteristic of blood that can affect analyte detection is thehaematocrit. Levels of haematocrit can be vastly different amongstvarious people. By way of non-limiting example, a person suffering fromanemia may have a haematocrit level of approximately 20% while a neonatemay have a haematocrit level of approximately 65%. Even samples takenfrom the same individual over a period of time can have differenthaematocrit levels. Further, because high haematocrit can also increasethe viscosity of blood, and viscosity can in turn affect otherparameters associated with analyte detection, accounting for the effectof haematocrit on a sample can be important in making accurate analyteconcentration determinations.

One way in which varying levels of haematocrit in a blood sample havebeen accounted for is by separating the plasma from the blood and thenrecalculating the concentration of the antigen with respect to theadjusted plasma volume. Separation has been achieved, for example, byperforming a centrifugation step. Other ways in which the varying levelsof haematocrit in a blood sample have been accounted for include usingan average haematocrit in a calculation or measuring a haematocrit in aseparate step and then calculating the concentration of the antigen withrespect to the plasma value. These methods, however, are believed to beundesirable, at least because they involve unwanted sample handling,take additional time, and/or lead to substantial errors in the finaldeterminations. Further, temperatures in environments where samples areanalyzed can also have a negative impact on the accuracy of analyteconcentration determination.

SUMMARY

Applicants have recognized that it would be desirable to develop a wayto obtain more accurate analyte concentration measurements that accountfor a wide spectrum of haematocrit levels and temperatures with littleor none of the attendant issues noted previously. Accordingly, systems,devices, and methods are generally provided for determining aconcentration of an analyte in a sample. In an exemplary embodiment of amethod for determining a concentration of an analyte in a sample, themethod includes detecting a presence of the sample in an electrochemicalsensor. The electrochemical sensor can include, for example, twoelectrodes. The two electrodes can include, for example, an opposedfaced orientation. In other embodiments, the two electrodes can includea facing orientation.

The method further includes determining a fill time of the sample withthe two electrodes and calculating a correction factor in view of atleast the fill time. The method also includes reacting an analyte tocause a physical transformation of the analyte between the twoelectrodes and determining the concentration of the analyte in view ofthe correction factor with the same two electrodes. For example,reacting of the analyte can generate an electroactive species that canbe measured as a current by the two electrodes. In some embodiments, thefill time determination and the analyte concentration determination canboth be determined using the same two electrodes.

In an exemplary embodiment of a method for measuring a corrected analyteconcentration, the method includes detecting a presence of the sample inan electrochemical sensor. The electrochemical sensor can include, forexample, two electrodes. The two electrodes can include, for example, anopposed faced orientation. In other embodiments, the two electrodes caninclude a facing orientation.

The method further includes determining a fill time of the sample withthe two electrodes. The method also includes reacting an analyte tocause a physical transformation of the analyte. The method furtherincludes determining a first analyte concentration in the sample withthe same two electrodes and calculating a corrected analyteconcentration based on the first analyte concentration and the filltime. In some embodiments, the fill time determination and the analyteconcentration determination can both be determined using the same twoelectrodes.

In one embodiment, the step of calculating the corrected analyteconcentration can include calculating a correction factor based on thefill time. In such an embodiment, the corrected analyte concentrationcan be calculated based on the first analyte concentration and thecorrection factor. In an exemplary embodiment, the correction factor canbe determined based on a series of threshold values. For example, thecorrection factor can be about zero when the fill time is less than afirst fill time threshold. For another example, the correction factorcan be calculated in view of the fill time when the fill time is greaterthan a first fill time threshold and less than a second fill timethreshold. For yet another example, the correction factor can be aconstant value when the fill time is greater than a second fill timethreshold.

In some embodiments, the details of the step of calculating thecorrected analyte concentration can depend on whether the first analyteconcentration in the sample is less than or greater than a thresholdvalue. For example, the step of calculating the corrected analyteconcentration can include a sum of the correction factor and the firstanalyte concentration in the sample when the first analyte concentrationin the sample is less than a threshold value. For another example, whenthe first analyte concentration in the sample is greater than athreshold value, the step of calculating the corrected analyteconcentration can include dividing the correction factor by one hundredand adding one to give an intermediate term and multiplying theintermediate term by the first analyte concentration to give a fill timecorrected analyte concentration.

In some embodiments of the above methods, the fill time of the samplecan be determined by applying an electric potential between the twoelectrodes while the sample is introduced, measuring cell current as afunction of time, and determining a current drop time based on cellcurrent as a function of time. In such an embodiment, the current droptime can correspond to the fill time of the sample. In some embodiments,the step of determining current drop time can include calculating themaximum negative value of the change in measured cell current over time.In some embodiments, the step of determining current drop time caninclude calculating a difference between at least two current valueswhere the difference is greater than a first predetermined threshold. Insome embodiments, the step of determining current drop time can includecalculating a difference between at least two current values where thedifference is less than a second predetermined threshold. In someembodiments, the step of determining current drop time can includecalculating a slope in the measured current as a function of time wherethe slope is greater than a third predetermined threshold. In someembodiments, the step of determining current drop time can includecalculating a slope in the measured current as a function of time wherethe slope is less than a fourth predetermined threshold. In someembodiments, the step of determining current drop time can includecalculating an inflection point in the measured current as a function oftime. The measurement of cell current as a function of time can include,for example, performing current measurements approximately every 2milliseconds and calculating and storing an average current based on thecurrent measurements approximately every 10 milliseconds. In someembodiments, the method can further include determining a level ofhaematocrit in the sample in view of the fill time of the sample. As aresult, the concentration of the antigen can be determined in view ofthe determined level of haematocrit.

In some embodiments of the above methods, detecting the presence of asample can include applying an electric potential between the twoelectrodes, and measuring a change in current values that is greaterthan a fifth predetermined threshold. In some embodiments, detecting thepresence of a sample can include applying an electric potential betweenthe two electrodes, and measuring a change in current values that isless than a sixth predetermined threshold. In some embodiments detectingthe presence of a sample can include applying a generally constantcurrent between the two electrodes and measuring a change in an electricpotential that is greater than a seventh predetermined threshold. Insome embodiments, detecting the presence of a sample can includeapplying a generally constant current between the two electrodes andmeasuring a change in an electric potential that is less than an eighthpredetermined threshold. In some embodiments, detecting the presence ofthe sample can be performed by a microprocessor of an analyte measuringmachine.

The electrochemical cell can include a glucose sensor. In anotherembodiment the electrochemical cell can include an immunosensor. In suchan embodiment, the analyte for which the concentration is being analyzedcan include C-reactive protein. The analyzed sample can include blood.In one embodiment the blood can include whole blood. The analyte forwhich the concentration is being analyzed can include glucose.

In an exemplary embodiment of a method for measuring a corrected analyteconcentration, the method includes detecting a presence of the sample inan electrochemical sensor. The electrochemical sensor can include, forexample, two electrodes. The method further includes determining a filltime of the sample with the two electrodes. The method also includesreacting an analyte that causes a physical transformation of theanalyte. The method further includes determining a first analyteconcentration in the sample with the same two electrodes and calculatinga corrected analyte concentration based on the first analyteconcentration and the fill time. In some embodiments, the fill timedetermination and the analyte concentration determination can both bedetermined using the same two electrodes.

In one embodiment, the step of calculating the corrected analyteconcentration can include calculating a correction factor based on thefill time. In such an embodiment, the corrected analyte concentrationcan be calculated based on the first analyte concentration and thecorrection factor. In an exemplary embodiment, the correction factor canbe determined based on a series of threshold values. For example, thecorrection factor can be about zero when the fill time is less than afirst fill time threshold. For another example, the correction factorcan be calculated in view of the fill time when the fill time is greaterthan a first fill time threshold and less than a second fill timethreshold. For yet another example, the correction factor can be aconstant value when the fill time is greater than a second fill timethreshold.

In some embodiments, the details of the step of calculating thecorrected analyte concentration can depend on whether the first analyteconcentration in the sample is less than or greater than a thresholdvalue. For example, the step of calculating the corrected analyteconcentration can include a sum of the correction factor and the firstanalyte concentration in the sample when the first analyte concentrationin the sample is less than a threshold value. For another example, whenthe first analyte concentration in the sample is greater than athreshold value, the step of calculating the corrected analyteconcentration can include dividing the correction factor by one hundredand adding one to give an intermediate term and multiplying theintermediate term by the first analyte concentration to give a fill timecorrected analyte concentration.

In one exemplary embodiment of an electrochemical system, the systemincludes an electrochemical sensor including electrical contactsconfigured to mate with a test meter. The electrochemical sensorincludes a first electrode and a second electrode in a spaced apartrelationship and a reagent. The first and second electrodes can include,for example, an opposed faced orientation. In other embodiments, thefirst and second electrodes can include a facing orientation. The systemalso includes a test meter including a processor configured to receivecurrent data from the test strip upon application of voltages to thetest strip, and further configured to determine a corrected analyteconcentration based on a calculated analyte concentration and a measuredfill time with the same two electrodes. The system can also include aheating element configured to heat at least a portion of theelectrochemical sensor. In some embodiments, the test meter can includedata includes data storage that contains an analyte concentrationthreshold, a first fill time threshold, and a second fill timethreshold. In some embodiments, at least one of the electrochemicalsensor, the test meter, and the processor are configured to measure atemperature of the sample.

In one embodiment, the electrochemical cell can be a glucose sensor. Inanother embodiment, the electrochemical cell can be an immunosensor. Theimmunosensor can include a first liquid reagent, a second liquidreagent, and magnetic beads conjugated to an antigen. In one embodimentthe first liquid reagent can include an antibody conjugated to an enzymein a buffer. The first liquid reagent can be striped on the lowerelectrode and can be dried. The second liquid reagent can includeferricyanide, a substrate for the enzyme, and a second mediator in adilute acid solution. The second liquid reagent can be striped on thelower electrode and can be dried. The magnetic beads, on the other hand,can be striped on the upper electrode and dried.

The immunosensor can also include a plurality of chambers, a separator,a vent, and one or more sealing components. The separator can bedisposed between the lower and the upper electrodes. The plurality ofchambers can include a reaction chamber, a detection chamber, and a fillchamber. The reaction chamber can be formed in the separator and canhave the first reagent and the magnetic beads conjugated to the antigendisposed therein. The detection chamber can also be formed in theseparator and can have the second reagent disposed therein. The fillchamber can be formed at least partially in the separator and one of thelower and upper electrodes, can be spaced a distance apart from thedetection chamber, and can overlap at least a portion of the reactionchamber. The vent can be formed at least partially in each of theseparator, the lower electrode, and the upper electrode, can be spaced adistance apart from the reaction chamber, and can overlap at least aportion of the detection chamber. In one embodiment the one or moresealing components can be a first sealing component and a second sealingcomponent. The first sealing component can have an incorporatedanticoagulant coupled to one of the lower and upper electrodes, can bedisposed over the vent, and can be configured to both form a wall of thefill chamber and seal the vent. The second sealing component can becoupled to the other of the lower and upper electrodes, can be disposedover the vent, and can be configured to seal the vent. In one embodimentthe first sealing component is a hydrophilic adhesive tape. At least oneof the control unit, the immunosensor, and the meter can include aconfiguration to measure a temperature of the sample. The analyte forwhich the system calculates the concentration can include C-reactiveprotein. The sample introduced into the electrochemical cell can includeblood. In one embodiment the blood can include whole blood.

The electrochemical sensor can also be a number of other analyzingdevices, including, by way of non-limiting example, electrochemicalcells, glucose sensors, glucose meters, hemoglobin sensors, antioxidantsensors, biosensors, and immunosensors. In one embodiment theelectrochemical sensor is a glucose sensor. The glucose sensor caninclude an electrochemical cell having a working electrode and a counteror counter/reference electrode. The working electrode and the counter orcounter/reference electrode can be spaced apart by approximately 500micrometers or less. In one embodiment a spacing between the electrodesis in the range of about 80 micrometers to about 200 micrometers. Thespacing can be determined in order to achieve a desired result, forexample, substantially achieving a steady state current in a desirabletime. In one embodiment a spacing between the electrodes is selectedsuch that the reaction products from a counter electrode arrive at aworking electrode.

The working and counter or counter/reference electrode can have avariety of configurations. For example, the electrodes can be facingeach other, they can be substantially opposed to each other, or they canhave a side-by-side configuration in which the electrodes are positionedapproximately in the same plane. The electrodes can have substantiallythe same corresponding area. The electrodes can also be planar. In oneembodiment the electrochemical cell includes a working electrode, acounter electrode, and a separate reference electrode. In anotherembodiment the electrochemical cell can have two electrode pairs. Theelectrode pairs can include any combination of working, counter,counter/reference, and separate reference electrodes, but in oneexemplary embodiment each pair includes a working electrode and acounter or counter/reference electrode. In still another embodiment theelectrochemical cell can have an effective cell volume of about 1.5microliters or less. The electrochemical cell can be hollow.

A potential can be applied to the electrodes of the cells by a number ofdifferent mechanisms, including, by way of non-limiting example, ameter. The magnitude of the potential can depend on a number ofdifferent factors, including, by way of non-limiting example, thedesired reaction of the sample within the cell. In one embodiment themagnitude of the potential can be selected such that electro-oxidationof a reduced form or electro-reduction of an oxidized form of a sampleis substantially diffusion controlled.

Samples can enter the cell by way of capillary action. A control unitcan be used to determine a fill time of the sample entering the cell. Inone embodiment the control unit can include a current flow detectorconfigured to measure cell current as a function of time to determine acurrent drop corresponding to the fill time of the sample. At least oneof the control unit, the electrochemical cell, and the meter can beconfigured to measure a temperature of the sample, or alternatively atemperature of the ambient air inside of the meter or proximate to theelectrochemical sensor attached to the meter.

One exemplary embodiment of a method for measuring an antigen in a bloodsample can include providing an immunosensor having two electrodes and ameter connected to the electrochemical cell so that the meter applies apotential between the two electrodes of the immunosensor. The method canfurther include introducing a blood sample including an antigen into theimmunosensor, applying an electric potential between the two electrodes,calculating a fill time of the blood sample, and determining aconcentration of the antigen in view of the fill time. The immunosensorcan further include a reaction chamber and a detection chamber formed ina separator disposed between the two electrodes, a fill chamber at leastpartially formed in the separator and one of the two electrodes, and avent at least partially formed in the separator and the two electrodes.The fill chamber can be spaced a distance apart from the detectionchamber and can overlap at least a portion of the reaction chamber. Thevent can be spaced a distance apart from the reaction chamber and canoverlap at least a portion of the detection chamber. The antigen of theblood sample can be C-reactive protein. The method can further includemeasuring a temperature of the blood sample. As a result, aconcentration of the antigen can be calculated in view of fill time.

The method for measuring a blood sample can further include providing anantibody-enzyme conjugate in a first buffer and magnetic beads linked toan antigen in a second buffer in the reaction chamber. Ferricyanide,glucose, and a mediator in a dilute acid can be provided in thedetection chamber. A first seal can be provided over a first side of thevent that forms a wall of the fill chamber and a second seal can beprovided over a second side of the vent. At least a portion of the bloodsample that is introduced into the immunosensor moves from the fillchamber to the reaction chamber when it is introduced into theimmunosensor.

The method can further include opening the vent after a pre-determinedtime by piercing at least one of the seals. Piercing at least one of theseals allows portions of the blood sample containing the antibody-enzymeconjugate that are not bound to the magnetic beads to move to thedetection chamber. Still further, the method can include catalyzingoxidation of the glucose in the detection chamber, which can result inthe formation of ferrocyanide. A current can be electrochemicallydetected from the ferrocyanide, and a concentration of the antigen inthe blood sample can be calculated in view of the signal detected.

BRIEF DESCRIPTION OF DRAWINGS

This invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 illustrates a flow chart of an exemplary method of a method ofdetermining the concentration of an analyte in a sample in accordancewith the present invention;

FIG. 2A illustrates a side elevation schematic drawing (not to scale) ofan exemplary embodiment of an electrochemical cell in accordance withthe present invention;

FIG. 2B illustrates a plan view, from above, of the electrochemical cellof FIG. 2A;

FIG. 3 illustrates a schematic drawing (not to scale), in cross-section,of an exemplary embodiment of a hollow electrochemical cell inaccordance with the present invention;

FIG. 4 A illustrates a perspective view of an assembled test inaccordance with the present invention;

FIG. 4B illustrates an exploded perspective view of an unassembled teststrip in accordance with the present invention;

FIG. 4C illustrates an expanded perspective view of a proximal portionof the test strip in accordance with the present invention;

FIG. 5A illustrates a bottom plan view of one embodiment of a test stripdisclosed herein;

FIG. 5B illustrates a side plan view of the test strip of FIG. 5A;

FIG. 5C illustrates a top plan view of the test strip of FIG. 5B;

FIG. 5D is a partial side view of a proximal portion of the test stripof FIG. 5C;

FIG. 6 illustrates an exploded view of an exemplary embodiment of animmunosensor in accordance with the present invention, wherein theimmunosensor is configured for use with a control unit having anelectrochemical detection system for calculating a fill time;

FIG. 7 illustrates a plot of a current versus time transient performedusing an exemplary embodiment of an electrochemical cell in conjunctionwith an exemplary embodiment for testing a variety of blood samplesprovided herein;

FIG. 8 illustrates a plot of a current versus time transient performedusing another exemplary embodiment of an electrochemical cell inconjunction with an exemplary embodiment for testing a variety of bloodsamples provided herein;

FIG. 9 illustrates a plot of the results of testing a variety of bloodsamples using a variable prepulse time method according to an exemplaryembodiment and a fixed time method;

FIG. 10 illustrates a plot of fill time versus haematocrit level for avariety of blood samples provided herein;

FIG. 11 illustrates a test voltage waveform in which the test meterapplies a plurality of test voltages for prescribed time intervals;

FIG. 12 illustrates a plot of the results of testing a variety of bloodsamples without correcting for fill time;

FIG. 13A illustrates the same data as FIG. 12 plotted against thehematocrit of the samples;

FIG. 13B illustrates a plot of the data shown in FIG. 12 corrected forfill time and plotted against the hematocrit of the sample;

FIG. 14 illustrates a plot of the results of testing a variety of bloodsamples in a clinical setting;

FIG. 15 illustrates a plot of current versus time transients obtainedwhen blood with hematocrits in the range of 15% to 72% was loaded intoanother exemplary embodiment of an electrochemical sensor in conjunctionwith an exemplary embodiment for testing a variety of samples providedherein.

FIG. 16 illustrates an alternate plot of the data shown in FIG. 15.

DETAILED DESCRIPTION

The following detailed description should be read with reference to thedrawings, in which like elements in different drawings are identicallynumbered. The drawings, which are not necessarily to scale, depictselected embodiments and are not intended to limit the scope of theinvention. The detailed description illustrates by way of example, notby way of limitation, the principles of the invention.

As used herein, the terms “about” or “approximately” for any numericalvalues or ranges indicate a suitable dimensional tolerance that allowsthe part or collection of components to function for its intendedpurpose as described herein. In addition, as used herein, the terms“patient,” “host,” “user,” and “subject” refer to any human or animalsubject and are not intended to limit the systems or methods to humanuse, although use of the subject invention in a human patient representsa preferred embodiment.

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the devices and methods disclosed herein. One ormore examples of these embodiments are illustrated in the accompanyingdrawings. Those skilled in the art will understand that the devices andmethods specifically described herein and illustrated in theaccompanying drawings are non-limiting exemplary embodiments and thatthe scope of the present invention is defined solely by the claims. Thefeatures illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present invention.

The presently disclosed systems and methods are suitable for use in thedetermination of a wide variety of analytes in a wide variety ofsamples, and are particularly suited for use in the determination ofanalytes in whole blood, plasma, serum, interstitial fluid, orderivatives thereof. In an exemplary embodiment, a glucose test systembased on a thin-layer cell design with opposing electrodes and tri-pulseelectrochemical detection that is fast (e.g., about 5 second or lessanalysis time), requires a small sample (e.g., about 0.4 μL or less),and can provide improved reliability and accuracy of blood glucosemeasurements. In the reaction cell to assay analyte, glucose in thesample can be oxidized to gluconolactone using glucose dehydrogenase andan electrochemically active mediator can be used to shuttle electronsfrom the enzyme to a palladium working electrode. More particularly, areagent layer coating at least one of the electrodes in the reactioncell can include glucose dehydrogenase (GDH) based on pyrroloquinolinequinone (PQQ) co-factor and ferricyanide. In another embodiment, theenzyme GDH based on the PQQ co-factor may be replaced with the enzymeGDH based on the flavin adenine dinucleotide (FAD) co-factor. When bloodor control solution is dosed into the reaction chamber, glucose isoxidized by GDH(ox) and in the process converts GDH(ox) to GDH(red), asshown in the chemical transformation T.1 below. Note that GDH(ox) refersto the oxidized state of GDH, and GDH (red) refers to the reduced stateof GDH.

D-Glucose+GDH(ox)−Gluconic acid+GDH(red)  T.1

A potentiostat can be utilized to apply a tri-pulse potential waveformto the working and counter electrodes, resulting in test currenttransients used to calculate the glucose concentration. Further,additional information gained from the test current transients may beused to discriminate between sample matrices and correct for variabilityin blood samples due to hematocrit, temperature variation,electrochemically active components, and identify possible systemerrors.

The subject methods can be used, in principle, with any type ofelectrochemical cell having spaced apart first and second electrodes anda reagent layer. For example, an electrochemical cell can be in the formof a test strip. In one aspect, the test strip may include two opposingelectrodes separated by a thin spacer for defining a sample-receivingchamber or zone in which a reagent layer is located. Applicants notethat other types of test strips, including, for example, test stripswith co-planar electrodes may also be used with the methods describedherein.

The methods for determining a concentration of an analyte in a sampledisclosed herein can be used with any sample analyzing device and/orsystem. The devices typically include at least one working electrode andone counter electrode between which an electric potential can beapplied. The sample analyzing device can generally be associated with acomponent for applying the electric potential between the electrodes,such as a meter. Applicants note that a variety of test meters can beused with the systems and methods described herein. However, in oneembodiment, the test meter includes at least a processor, which mayinclude one or more control units configured for performing calculationscapable of calculating a correction factor in view of at least onemeasured or calculated parameter as well as configured for data sortingand/or storage. The microprocessor can be in the form of a mixed signalmicroprocessor (MSP) such as, for example, the Texas Instruments MSP430. The TI-MSP 430 can be configured to also perform a portion of thepotentiostat function and the current measurement function. In addition,the MSP 430 can also include volatile and non-volatile memory. Inanother embodiment, many of the electronic components can be integratedwith the microcontroller in the form of an application specificintegrated circuit.

The sample analyzing device can also be associated with one or morecomponents that are capable of measuring a fill time of a sample when itis introduced to the device. Such components can also be capable ofcalculating a concentration of an analyte in the sample in view of thefill time. Such components are generally referred to herein as controlunits. Further, the terms analyte, antigen, and antibodies are usedinterchangeably within, and thus, use of one term is equally applicableto all three terms, unless otherwise indicated or reasonably known byone skilled in the art.

In one exemplary embodiment of a method for determining a concentrationof an analyte in a sample, a sample is introduced into anelectrochemical cell of a sample analyzing device that has a workingelectrode and a counter electrode. An electric potential can be appliedbetween the working and counter electrodes of the electrochemical celland a fill time of the sample into, for example, a capillary space ofthe electrochemical cell, can be determined. A prepulse time can becalculated in view of at least the fill time of the sample and anelectric potential can be applied between the working electrode and thecounter electrode for a length of time equal to the prepulse time. Aconcentration of the analyte in the sample can then be determined. Bycalculating the prepulse time in view of the fill time, more accurateresults can be achieved for analyte concentration. For example, errors,such as those that can result from varying haematocrit levels acrosssamples, can be accounted for, thereby leading to more accuratedeterminations of the concentrations of the analytes in the samples.Methods can also account for the effects of temperature, as discussed ingreater detail below. In an alternative embodiment for detecting aconcentration of an analyte in a sample, errors are corrected for basedon a determined initial fill velocity rather than a determined filltime. One example of such a method is disclosed in U.S. patentapplication Ser. No. 12/649,509 entitled “Systems, Devices and Methodsfor Measuring Whole Blood Haematocrit Based on Initial Fill Velocity,”of Ronald C. Chatelier, Dennis Rylatt, Linda Raineri, and Alastair M.Hodges, and filed on Dec. 30, 2009, the contents of which is herebyincorporated by reference in its entirety.

In an alternative embodiment, an estimate of a level of haematocritlevel can be determined. In some embodiments, the estimate of a level ofhaematocrit can be determined without reference to an associated analyteconcentration. As a result, assessments related to conditions such asanemia can be made. In such a system, only a level of haematocrit ismeasured without making other concentration determinations. Determininga level of haematocrit based on the disclosed teachings can allowdeterminations to be made quickly and accurately, often in less than asecond. For example, haematocrit levels of a drop of blood can bedetermined in less than a second merely by dropping the blood onto asensor strip of a sample analyzing device. Once the blood is disposed onthe strip, a digital readout of the haematocrit level can be providedalmost instantaneously.

A fill time can be used in a variety of ways to improve a determinationof a concentration of an analyte. For example, the fill time of thesample can be used to calculate a prepulse time. By adjusting theprepulse time in view of the fill time, longer reaction times can beprovided for samples which take a longer time to fill the sensor. Forexample, if the sample includes whole blood, then haematocrit level canbe a factor in the fill time of the sample. Adjusting the prepulse timein view of the fill time can thus allow for more accurate concentrationsto be determined over a range of haematocrit levels. In someembodiments, the haematocrit level can be linked to the fill time, e.g.,an estimate of the haematocrit level can be determined in view of thefill time. In such an instance, the haematocrit levels can be accountedfor in the determination of the analyte concentration in order toprovide more accurate analyte concentration determinations.

In one exemplary embodiment, the steps illustrated in FIG. 1 can be usedto determine the concentration of an analyte in a sample. As shown, asample is first introduced into the device. Any type of sample analyzingdevices can be used in conjunction with at least some of the systems andmethods disclosed herein. These devices can include, by way ofnon-limiting example, electrochemical cells, electrochemical sensors,glucose sensors, glucose meters, hemoglobin sensors, antioxidantsensors, biosensors, and immunosensors. One exemplary embodiment of asample analyzing device is an electrochemical sensor. Theelectrochemical sensor can include at least two electrodes. The at leasttwo electrodes can be configured in any way, for example, the electrodescan be on the same plane or on different planes. A sample can beintroduced into the electrochemical cell.

In one embodiment, the introduction of a sample may be detected by anautomatic technique in which the meter monitors a change in voltage,current, or capacitance, a change which indicates that sample has beendosed into the sample reaction chamber. Alternatively, the physiologicalsample may be detected by a manual technique in which the user visuallyobserves the filling of the sample reaction chamber and initiates thetest by pressing a button. In another embodiment, an optical detector inthe meter can sense the dosing of the sample. The time taken by thesample to fill the reaction chamber can likewise be measured by anynumber of similar techniques. In one embodiment, the electrodes can beconfigured such that when a sample is introduced into the sensor, thesecond electrode is contacted prior to or simultaneous with the firstelectrode as the sample fills the sensor. However, as the sample fillsthe sensor, the first electrode is limiting in the current it cansustain relative to the voltage applied to the second electrode. Thefirst electrode can therefore limit the current flowing in theelectrochemical sensor. Prior to, simultaneous with, or immediatelyafter the sample contacts the first electrode, a potential can beapplied between the electrodes such that when the first and secondelectrodes are bridged by the sample liquid a current flows betweenthem. In one embodiment of the methods disclosed herein, the currentversus time response during the sensor filling can be used to determinethe point at which the sensor is adequately filled. For example,adequate filling can mean that sufficient liquid has filled the sensorto entirely cover at least the first electrode. In some embodiments, thecurrent versus time response can be a discontinuity in the rate ofchange of current with time, such as an increased drop in current or adecreased rate of increase. One example of the above methods isdisclosed in U.S. patent application Ser. No. 12/885,830 of Kranendonket al., entitled “Apparatus and Method for Improved Measurements of aMonitoring Device,” and filed on Sep. 20, 2010, the contents of which ishereby incorporated by reference in its entirety.

In one embodiment of the methods disclosed herein, a potential ofbetween about +10 mV to about +30 mV can be applied between the firstand second electrodes of an electrochemical cell for a period of time,e.g., about 1000 ms, as a sample introduced into the device fills thecell. In one exemplary embodiment, a potential of about +20 mV can beapplied between the first and second electrodes as a sample introducedinto the device fills the cell. The current flowing between theelectrodes can be measured at predetermined intervals during this time.For example, the current can be measured every 2 milliseconds (“ms”) andthe average current can be stored every 10 ms. The current data can thenbe analyzed, by a control unit, for example. In some embodiments, thecontrol unit can include a microprocessor. The analysis of the currentdata measured over the approximately 1000 ms, during which the samplefills the device, can include a determination of the latest time atwhich the current decreases by a predetermined amount. This time can beused as the fill time (FT) of the sample. For example, in oneembodiment, the latest time at which the current decreases by more than0.4 micro-Ampere (“μA”) over a 40 ms interval can be used to determinethe time at which the sample has filled the cell.

In some embodiments, the step of determining current drop time caninclude calculating a difference between in at least two current valueswhere the difference is greater than or less than a predeterminedthreshold value. Various predetermined threshold values can be employed.For example, when the area of the working electrode is about 4.2 squaremillimetres and hematocrits as high as about 75% are being assayed, thepredetermined threshold value can be in the range of about 0.4micramperes over about a 40 ms time period. In other exemplaryembodiment, when the area of the working electrode is about 4.2 squaremillimetres and hematocrits as high as about 60% are being assayed, thepredetermined threshold value can be in the range of about 0.7microamperes to about 0.9 micramperes over about a 50 ms time period. Insome embodiments, the step of determining current drop time can includecalculating an inflection point in the measured current as a function oftime.

In some embodiments, detecting the presence of a sample can includeapplying an electric potential between the two electrodes, and measuringa change in current values that is greater than or less than apredetermined threshold value. Various predetermined threshold valuescan be employed. For example, when the area of the working electrode isabout 4.2 square millimeters, the predetermined threshold value can bein the range of about 3 microamperes. In other embodiments, detectingthe presence of a sample can include applying a generally constantcurrent between the two electrodes, and measuring a change in anelectric potential that is greater than or less than a predeterminedthreshold. For example, the predetermined threshold value can be in therange of about 200 mV. In other exemplary embodiment, the thresholdvalue can be about 400 mV.

After the sample has filled the cell, a first electric potential, havinga first polarity, can be applied between a first and second electrodeand a resulting current measured as a function of time. This firstelectric potential can be referred to, for example, as a prepulse. Insome embodiments, the length of time that a prepulse can be applied canbe about 5 seconds. In other embodiments, the fill time (FT) of thesample, which can be determined using any of the techniques discussedabove, can be used to calculate the length of time that a prepulse canbe applied. This time period can be referred to, for example, as aprepulse time (PPT). For example, the calculation of prepulse time canallow for longer prepulse times for samples that take longer to fill thesensor. In one embodiment, the prepulse time can be set according to thefollowing exemplary parameters. For example, the prepulse time can becalculated as:

PPT(ms)=3000+(FT−300)×9.3

For purposes of this calculation, for fill times less than 300 ms, thefill time can be set to 300 ms. This calculation allows the prepulsetime (PPT) to be adjusted to allow for longer reaction times for samplesthat take more than a predetermined amount of time, e.g., about 300 ms,to fill the sensor. For purposes of simplifying calculation and to placeboundaries on the total test time a maximum prepulse time can be set ifthe fill time is longer than a predetermined length of time. Forexample, in one embodiment, if the fill time is greater than about 500ms, e.g., about 515 ms, the prepulse time (PPT) can be set equal to 5000ms. Thus, in this exemplary embodiment, the minimum PPT (for fill timesless than about 300 ms) is 3000 ms and the maximum PPT (for fill timesgreater than about 500 ms, e.g., about 515 ms) is about 5000 ms. Inother embodiments, the calculation of prepulse time can be adjusted soas to take into account other properties and requirements of aparticular sample or analyte. For example, the variables and constantsin the equation shown above for calculation of prepulse time can beadjusted so as to provide alternate maximum and minimum prepulse times,or combinations thereof.

Once the prepulse time has been determined, a potential can be appliedbetween the electrodes of the cell for a time equal to the prepulse time(PPT) and a resulting current measured as a function of time. At least aportion of the data (current as a function of time) provides a firsttime-current transient. The first electrical potential can besufficiently negative with respect to the second electrode such thatsecond electrode functions as the working electrode in which a limitingoxidation current is measured. After the first time interval haselapsed, a second electric potential can be applied between the firstand second electrodes for a second time interval. The second electricalpotential causes a current that is measured as a function of time toproduce a second time-current transient. In one embodiment, the secondpotential has a second polarity, which is opposite to the firstpolarity. For example, the second potential can be sufficiently positivewith respect to second electrode such that first electrode functions asthe working electrode in which a limiting oxidation current is measured.In one exemplary embodiment, the first electric potential and secondelectrical potential can range from about −0.6 V to about +0.6 V. Thetime interval of the time-current transients can, in one embodiment, canbe in the range of about 1 second to 10 seconds, and preferably in therange of about 1 to 5 seconds. In another embodiment, a sum of the firsttime interval and the second time interval is less than about 5 seconds.It should also be noted that the first time interval does not have to bethe same as the second time interval. In one embodiment, the secondelectric potential is applied immediately following the application ofthe first electric potential. In an alternative embodiment, a delay oropen circuit potential is introduced in between the first electricpotential and the second electric potential. In another alternativeembodiment, a delay is introduced after physiological sample is detectedin the sample reaction chamber, but before the application of the firstelectric potential. The delay can be in the range of about 0.01 andabout 3 seconds, preferably from about 0.05 to about 1 second and mostpreferably from about 0.5 to about 0.9 seconds.

In one exemplary embodiment, a first test potential E₁ can be appliedbetween the electrodes for a first test potential time T₁, e.g., PPTmilliseconds. For example, a potential of +300 mV can be applied. Afterthe first test potential time T₁, e.g., PPT milliseconds, has elapsed, asecond test potential E₂ can be applied between the electrodes for asecond test potential time interval T₂, e.g., −300 mV for 1000 ms.During T₁ and T₂, the cell current as a function of time can bemeasured, herein called a time current transient or a current transientand referred to as i_(a)(t), during first test potential time intervalT₁, and as i_(b)(t) during the second test potential time interval T₂.For example, the current as a function of time can be measured every 10ms with the average current stored every 50 ms. At least a portion ofthe data from the first and second potentials (current as a function oftime) can provide first and second time-current transients. Theconcentration of an analyte in the sample can then be determined fromthe current data using any number of algorithms.

Examples of algorithms for determining analyte concentration can befound at least in U.S. patent application Ser. No. 11/278,341 ofChatelier et al., entitled “Methods And Apparatus For Analyzing A SampleIn The Presence Of Interferents,” and filed on Mar. 31, 2006, thecontents of which is hereby incorporated by reference in its entirety.In one exemplary embodiment, the current data can be analyzed using a“calibration-free, corner-corrected algorithm” similar to thosedisclosed in the aforementioned patent application. In one embodiment,an analyte concentration can be calculated using the algorithm as shownin Equation (Eq.) 1.

$\begin{matrix}{G = {\left( \frac{i_{r}}{i_{l}} \right)^{\gamma}\left\{ {{ai}_{2} - {zgr}} \right\}}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

In Eq. 1, G is the analyte concentration, the terms i_(l), i_(r) and i₂are current values and the terms p, zgr, and a are empirically derivedcalibration constants.

In one embodiment of the invention, p may range from about 0.2 to about4, and preferably from about 0.1 to about 1. The calibration factor acan be used to account for possible variations in the dimensions of theelectrochemical cell. Variations in the dimensions of theelectrochemical cell can cause a proportional shift in the magnitude ofthe measured current. Under certain circumstances, manufacturingprocesses can cause the electrode area to vary from one lot of teststrips to another lot of test strips. Calculating a calibration factor afor each lot of test strips helps to compensate for variations inelectrode area and the height of the cell. The term a can be calculatedduring the calibration process of a test strip lot.

A calibration factor zgr is used to account for variations in thebackground. A presence of an oxidizable species within the reagent layerof the cell before the addition of a sample may contribute to abackground signal. For example, if the reagent layer were to contain asmall amount of ferrocyanide (e.g., reduced mediator) before the samplewas added to the test strip, then there would be an increase in themeasured test current which would not be ascribed to the analyteconcentration. Because this would cause a constant bias in the overallmeasured test current for a particular lot of test strips, this bias canbe corrected for using the calibration factor Z. Similar to the terms pand a, Z can also be calculated during the calibration process.Exemplary methods for calibrating strip lots are described in U.S. Pat.No. 6,780,645 which is hereby incorporated by reference in its entirety.

In one exemplary embodiment, p can be 0.51, a can be 0.2, and zgr can be5. While the method disclosed herein is described with the use ofcalibration factors, p, a, and zgr, one skilled in the art willappreciate that their use is not required. For example, in oneembodiment, glucose concentration could be calculated without p, a,and/or Z (in Eq. 1 p and/or a could be set equal to one and zgr could beset equal to zero). A derivation of Eq. 1 can be found in a pending U.S.application Ser. No. 11/240,797 which was filed on Sep. 30, 2005 andentitled “Method and Apparatus for Rapid Electrochemical Analysis,”which is hereby incorporated by reference in its entirety.

Current value i_(r) can be calculated from the second current transientand current value i_(l) can be calculated from the first currenttransient. All current values (e.g. i_(r), i_(l), and i₂) stated in Eq.1 and in subsequent equations can use the absolute value of the current.Current values i_(r), i_(l), can be, in some embodiments, an integral ofcurrent values over a time interval of a current transient, a summationof current values over a time interval of a current transient, or anaverage or single current value of a current transient multiplied by atime interval of the current transient. For the summation of currentvalues, a range of consecutive current measurement can be summedtogether from only two current values or to all of the current values.Current value i₂ can be calculated as discussed below.

For example, where the first time interval is 5 seconds long, i_(l) maybe the average current from 1.4 to 4 seconds of a 5 second long periodand i_(r) may be the average current from 4.4 to 5 seconds of a 5 secondlong period, as shown in Eq. 2a and 3a, below.

$\begin{matrix}{i_{r} = {\sum\limits_{t = 4.4}^{5}{i(t)}}} & {{{Eq}.\mspace{14mu} 2}a} \\{i_{l} = {\sum\limits_{t = 1.4}^{4}{i(t)}}} & {{{Eq}.\mspace{14mu} 3}a}\end{matrix}$

In another example, where the first interval is 5 seconds long i_(l) maybe the sum of currents from 3.9 to 4 seconds of a 5 second long periodand i_(r) may be the sum of currents from 4.25 to 5 seconds of a 5second long period, as shown in Eq. 2b and 3b, below.

$\begin{matrix}{i_{r} = {\sum\limits_{t = 4.25}^{5}{i(t)}}} & {{{Eq}.\mspace{14mu} 2}b} \\{i_{l} = {\sum\limits_{t = 3.9}^{4}{i(t)}}} & {{{Eq}.\mspace{14mu} 3}b}\end{matrix}$

A magnitude of current for the first current transient can be describedas a function of time by Eq. 4.

$\begin{matrix}{{i_{a}(t)} = {i_{ss}\left\{ {1 + {2{\sum\limits_{n = 1}^{\infty}{\exp\left( \frac{{- 4}\pi^{2}n^{2}{Dt}}{L^{2}} \right)}}}} \right\}}} & {{Eq}.\mspace{14mu} 4}\end{matrix}$

The term i_(ss) is the steady-state current following the application offirst test potential E₁, D is the diffusion coefficient of the mediator,L is the thickness of the spacer. It should be noted that in Eq. 4, trefers to the time elapsed after first test potential E₁ was applied. Amagnitude of current for the second current transient can be describedas a function of time by Eq. 5.

$\begin{matrix}{{i_{b}(t)} = {i_{ss}\left\{ {1 + {4{\sum\limits_{n = 1}^{\infty}{\exp\left( \frac{{- 4}\pi^{2}n^{2}{Dt}}{L^{2}} \right)}}}} \right\}}} & {{Eq}.\mspace{14mu} 5}\end{matrix}$

There is a factor of two difference for the exponential term in Eq. 5 ascompared to the exponential term in Eq. 4 because the second currenttransient is generated from the second test potential E₂, which wasopposite in polarity to the first test potential E₁, and was appliedimmediately after the first test potential E₁. It should be noted thatin Eq. 5, t refers to the time elapsed after second test potential E₂was applied.

A peak current for first test potential time interval T₁ can be denotedas i_(pa) and a peak current for second test potential time interval T₂can be denoted as i_(pb). If both first peak current i_(pa) and secondpeak current i_(pb) were measured at the same short time after theapplication of first test potential E₁ and second test potential E₂respectively, for example 0.1 seconds, Eq. 4 can be subtracted from Eq.5 to yield Eq. 6.

i _(pb)−2i _(pa) =−i _(ss)  Eq. 6

Because it has been determined that i_(pa) is controlled mainly byinterferents, i_(pb) can be used with i_(pa) together to determine acorrection factor. For example, as shown below i_(pb) can be used withi_(pa) in a mathematical function to determine a corrected current whichis proportional to glucose and less sensitive to interferents.

Eq. 7 was derived to calculate a current i₄ which is proportional toglucose and has a relative fraction of current removed that is ascribedto interferents.

$\begin{matrix}{i_{2} = {i_{r}\left\{ \frac{i_{pb} - {2i_{pa}} + i_{ss}}{i_{pb} + i_{ss}} \right\}}} & {{Eq}.\mspace{14mu} 7}\end{matrix}$

The term i_(ss) was added to both the numerator and denominator to allowthe numerator to approach zero when no glucose is present. The termi_(ss) may be estimated using Equation 8A, for currents at times greaterthan a minimum time, where a suitable minimum time can be estimated fromEquation 8B.

$\begin{matrix}{{i(t)} = {i_{ss}\left\{ {1 + {4{\exp\left( \frac{{- 4}\pi^{2}{Dt}}{L^{2}} \right)}}} \right\}}} & {{{Eq}.\mspace{14mu} 8}A} \\{t_{m\; i\; n} = \frac{{- L^{2}}\ln \; 0.01}{12\pi^{2}D}} & {{{Eq}.\mspace{14mu} 8}B}\end{matrix}$

in which, i_(ss) is the steady-state current following application ofthe second electric potential; i is the measured current which is afunction of time; D is the diffusion coefficient of the redox-activemolecule, where this coefficient may be determined from Fick's firstlaw, i.e. J(x,t)=−DdC(x,t)/dx; L is the spacer thickness; and t is thetime for the application of the second electric potential where t=0 forthe beginning of the second time interval.

In one exemplary embodiment, the current value, i₂, can be calculatedaccording to Eq. 9.

$\begin{matrix}{i_{2} = {i_{r}\left( \frac{{i(4.1)} - {2{i(1.1)}} + i_{ss}}{{i(4.1)} + i_{ss}} \right)}} & {{Eq}.\mspace{14mu} 9}\end{matrix}$

Thus, Eq. 1 can enable accurate measurements of analyte concentration inthe presence of interferents.

As discussed above, an estimate of a level of haematocrit can bedetermined without reference to an associated analyte concentration. Forexample, haematocrit levels of a drop of blood can be determined fromcurrent values and an analyte concentration. In one exemplaryembodiments, an estimate of the haematocrit (H) can be derived from Eq.10.

H=−162.5 log(i _(r))+119.1 log(G)+235.4  Eq. 10

In some embodiments, the value of the analyte concentration (G) can becorrected in view of the haematocrit level, e.g., using Eq. 11A and 11B.

G′=G+Corr for G<100 mg/dL  Eq. 11A

G′=G(1+Corr/100) for G≧100 mg/dL  Eq. 11B

In Eq. 11A and 11B, the correction factor Corr can be calculated usingsine functions whose amplitude varies with H. For example, at values ofH<30% the following equations can be used to calculate Corr.

Corr=−0.4(30−H)sin(πG/400) for G<400 mg/dL  Eq. 12A

Corr=0 for G≧400 mg/dL  Eq. 12B

where the range of Corr is restricted to 0 to −5.

When H>50%, an “asymmetric sine function” can be used where theamplitudes of the positive and negative lobes are different. However,the function is continuous so that there is no sudden step in thecorrection. For example, Eq. 13A to 13C can be used to calculate Con forH>50%.

Corr=−0.2(H−50)sin(πG/180) for G<180 mg/dL  Eq. 13A

Corr=−0.5(H−50)sin(πG/180) for 180≦G≦270 mg/dL  Eq. 13B

Corr=+0.5(H−50) for G>270 mg/dL  Eq. 13C

where the range of Corr is restricted to 0 to −5 for G≦180, and 0 to 5for G≧180.

In another embodiment, the value of the analyte concentration (G) can becorrected in view of the fill time without deriving an estimate of thehaematocrit (H), e.g., using Eq. 14A (when G<100 mg/dL) and 14B (whenG≧100 mg/dL) in conjunction with Eqs. 15A, 15B, and 15C.

G′=G+Corr for G<100 mg/dL  Eq. 14A

G′=G(1+Corr/100) for G≧100 mg/dL  Eq. 14B

The correction factor Corr in Eq. 14A and 14B can be calculated in viewof the fill time (FT) based on a series of threshold values of FT. Forexample, the following equations can be used to calculate Corr using twothreshold values of FT, Th₁ and Th₂.

if Th ₁<FT<Th ₂ then Corr=50(FT−Th ₁)  Eq. 15A

if FT<Th₁ then Corr=0  Eq. 15B

if FT>Th₂ then Corr=10  Eq. 15C

In an exemplary embodiment, the threshold value Th₁ can be about 0.2seconds and the threshold value Th₂ can be about 0.4 seconds. Forexample, when blood fills the sensor in less than about 0.2 seconds,then its fill behavior can be described as close to ideal. Fill times ofless than about 0.2 seconds usually occur when the hematocrit is lowenough that that the viscosity of the sample has a minimal effect on thefill behavior of the sample. As a consequence of the low hematocrit,most of the glucose is believe to be partitioned into the plasma phasewhere it can be oxidized rapidly. Under these conditions, there islittle need to correct the glucose result for the effect of fill time,and so the correction factor can be set to zero. Alternatively, when thehematocrit in the sample is high, the viscosity of the sample can affectthe fill time of the sample. As a results, the sample can take more thanabout 0.4 seconds to fill the sensor. As a consequence of the highhematocrit, most of the glucose is believe to be partitioned into thered blood cells and so a lower fraction of the glucose is oxidized.Under these conditions, the glucose result can be corrected in view ofthe fill time. However, it can be important not to over-correct theglucose value, and so, in an exemplary embodiment, the correction factorcan be restricted to a maximum of about 10 mg/dL plasma glucose or about10% of the signal. An empirically-derived linear equation can be used togradually increase the correction term in the range of about 0 to about10 as the fill time increases in the range of about 0.2 to about 0.4seconds.

One exemplary embodiment of a device that can be used in conjunctionwith at least some of the systems and methods disclosed herein is aglucose sensor. The glucose sensor can include an electrochemical cell,such as the cell illustrated in FIGS. 2A and 2B. The cell can include athin strip membrane 201 having upper and lower surfaces 202, 203, andcan also include a cell zone 204 defined between a working electrode 206disposed on the lower surface 203 and a counter/reference electrode 205disposed on the upper surface 202. The membrane thickness can beselected to achieve a desired result, such as having the reactionproducts from a counter electrode arrive at a working electrode. Forinstance, the membrane thickness can be selected so that the electrodesare separated by a distance t, which can be sufficiently close such thatthe products of electrochemical reaction at the counter electrode canmigrate to the working electrode during the time of the test and asteady state diffusion profile can be substantially achieved. Typicallyt can be less than approximately 500 micrometers, alternatively in therange of about 10 micrometers to about 400 micrometers, and moreparticularly in the range of about 80 micrometers to about 200micrometers. In one embodiment a spacing between the electrodes can beselected such that the reaction products from a counter electrode arriveat a working electrode before the end of the assay.

The electrodes can also have a variety of configurations. For instance,the electrodes can be planar. Further, while in the illustratedembodiment the electrodes 205, 206 are facing each other and aresubstantially opposed, in other embodiments the electrodes can just befacing each other, they can be substantially opposed to each other, orthey can have a side-by-side configuration in which the electrodes arepositioned approximately in the same plane. Examples of differentelectrode configurations can be found at least in U.S. Pat. No.7,431,820 of Hodges, entitled “Electrochemical Cell,” and filed on Oct.14, 2003, the contents of which is hereby incorporated by reference inits entirety.

A sample deposition or “target” area 207 can be defined on the uppersurface 202 of the membrane 201 and can be spaced at a distance greaterthan the membrane thickness from the cell zone 204. The membrane 201 canhave a diffusion zone 208 that can extend between the target area 207and the cell zone 204. A suitable reagent can include a redox mediatorM, an enzyme E, and a pH buffer B, each of which can be contained withinthe cell zone 204 of the membrane and/or between the cell zone 204 andthe target area 207. The reagent can also include stabilizers and thelike. In use of the sensor, a drop of blood can be placed on the targetzone 207, and the blood components can wick towards the cell zone 204.

Each of electrodes 205, 206 can have a predefined area. In theembodiments of FIGS. 2A and 2B the cell zone 204 can defined by edges209, 210, 211 of the membrane, which can correspond with edges of theelectrodes 205, 206 and by leading (with respect to the target area 207)edges 212, 213 of the electrodes. In the present example the electrodescan be about 600 angstrom thick and can be from about 1 to about 5 mmwide although a variety of other dimensions and parameters can be usedwithout departing from the scope of the present invention.

Alternatively, both sides of the membrane can be covered with theexception of the target area 207 by laminating layers 214 (omitted fromplan views) which can serve to prevent evaporation of water from thesample and to provide mechanical robustness to the apparatus.Evaporation of water is believed to be undesirable as it concentratesthe sample, allows the electrodes to dry out, and allows the solution tocool, affecting the diffusion coefficient and slowing the enzymekinetics, although diffusion coefficient can be estimated as above.

In an alternative embodiment, illustrated in FIG. 3, a hollowelectrochemical cell for use with the systems and methods disclosedherein is provided. The electrodes 305, 306 can be supported by spacedapart polymer walls 330 to define a hollow cell. An opening 331 can beprovided on one side of the cell whereby a sample can be admitted intothe cavity 332. In this embodiment a membrane is not used, although insome embodiments a membrane can be included. The electrodes can have avariety of configurations, at least as discussed above. By way ofnon-limiting example, the electrodes can be spaced apart by less thanabout 500 micrometers, preferably in the range of about 10 or about 20micrometers to about 400 micrometers, and more preferably in a range ofabout 80 micrometers to about 200 micrometers. The effective cell volumecan be about 1.5 microliters or less.

The electrochemical cells of FIGS. 2A, 2B, and 3 can be used inconjunction with the meters, control units, and other components andsteps of the devices, systems, and methods disclosed herein. Furtherdisclosures related to the electrochemical cells of FIGS. 2A, 2B, and 3are found in U.S. Pat. No. 6,284,125 of Hodges et al., entitled“Electrochemical cell” and filed on Apr. 17, 1998, the contents of whichis hereby incorporated by reference in its entirety. For example,electrochemical cells used in conjunction with the present disclosurescan have two electrode pairs. The electrode pairs can include anycombination of working, counter, counter/reference, and separatereference electrodes.

Another exemplary embodiment of a device that can be used in conjunctionwith at least some of the systems and methods disclosed herein is thesensor described below and illustrated in FIGS. 4A through 5D. Thesensor can be in the form of a form of a test strip 62 including anelongate body 59 that extends along a longitudinal axis L from aproximal end 80 to a distal end 82 and having lateral edges 56, 58. Body59 can include a proximal sample reaction chamber 61 that containselectrodes 164, 166 and a reagent 72. Test strip body 59 can furtherinclude distally positioned electrical contacts 63, 67 for electricallycommunicating with a test meter (not illustrated).

In one aspect, test strip 62 is formed from multiple layers including afirst electrically conductive layer 66, a spacer 60, a secondelectrically conductive layer 64. First electrically conductive layer 66and/or second electrically conductive layer 64 can be formed from avariety a conductive materials that are, in one embodiment, positionedon an insulating sheet (not shown). Spacer layer 60 can be formed from avariety of electrically insulating materials and can include, or beformed from, an adhesive. One skilled in the art will appreciate thatwhile a three layer test strip is illustrated, additional electricallyconductive or insulative layers could be used to form test strip body59.

As illustrated in FIGS. 4A through 4C, proximal sample reaction chamber61 can be defined by first electrically conductive layer 66, secondelectrically conductive layer 64, and spacer layer 60. As discussed inmore detail below, reaction chamber 61 can also include a reagent 72 andfirst and second electrodes 166, 164. For example, a cutout area 68 inspacer 60 can expose a portion of second electrically conductive layer64 and first electrically conductive layer 66, and thereby defines firstelectrode 166 and second electrode 164, respectively. Reagent 72 can bein the form of a layer positioned on first electrode 166.

In one embodiment, reaction chamber 61 is adapted for analyzing smallvolume samples. For example, sample reaction chamber 61 can have avolume ranging from about 0.1 microliters to about 5 microliters,preferably about 0.2 to about 3 microliters, and more preferably about0.3 microliters to about 1 microliter. To accommodate a small samplevolume, the electrodes are preferably closely spaced. For example, wherespacer 60 defines the distance between first electrode 166 and secondelectrode 164, the height of spacer 60 can be in the range of about 1micron to about 500 microns, preferably in the range of about 10 micronsand about 400 microns, and more preferably in the range of about 40microns and about 200 microns.

To further assist with the reduction in the volume of reaction chamber61 the longitudinal and/or lateral dimension of cutout area 68 and/orbody 59 can be adjusted. For example, test strip body 59 can includecut-away portions 51, 52 such that the lateral width of reaction chamber61 is smaller than the full width (widest width) of test strip body 59.Cut-away portions 51, 52 can also facilitate delivery of a sample toreaction chamber 61. For example, cut-away portion 51, 52 can have ashape corresponding to a portion of a finger of a user. When a userexpresses a drop of blood with a finger stick, the cut-away portions 51,52 can help the user align a sample positioned on his/her finger with asample receiving port (e.g., openings 70) in the lateral edge 56, 58 ofbody 59. One skilled in the art will appreciate that while two cut-awayportions are illustrated, test strip body 59 could include only a singlecut-away portion or no cut-away portions.

As stated above, the proximal portion of test strip body 59 can includeat least one sample delivery port for delivery of a sample to reactionchamber 61. For example, cutout area 68 can extend transversely to thelateral edges 56, 58 of test strip body 59 to provide two openings 70for the delivering of physiological fluid to sample reaction chamber 61.Where two openings 70 are present one can act as a sample receiving portfor delivery of a fluid sample while the other can act as a vent. Oneskilled in the art will appreciate that sample can be delivered tosample reaction chamber 61 using alternative structures including samplereceiving ports and/or vents positioned at different locations in teststrip body 59, such as, for example, sample receiving ports and/or ventspositioned in first and/or second electrically conductive layers 66, 64.

In one embodiment, test strip 62 is adapted to draw sample into reactionchamber 61 via capillary action. For example, the dimensions and surfacecharacteristics of reaction chamber 61 and openings 70 can be adapted toproduce a capillary force when a liquid sample (e.g., whole blood) isbrought into contact with one of openings 70. One skilled in the artwill appreciate that reaction chamber 61 can include additionalstructures to assist with/create capillary forces such as, for example,beads, a porous membrane, and/or other fillers.

As mentioned above, a reagent, such as reagent 72, can be disposedwithin reaction chamber 61. The composition of reagent 72 can varydepending on the intended analyte and the expected form of the sample.In one aspect, reagent 72 includes at least a mediator and an enzyme andis deposited onto first electrode 166. Various mediators and/or enzymesare within the spirit and scope of the present disclosure. For examplesuitable mediators include ferricyanide, ferrocene, ferrocenederivatives, osmium bipyridyl complexes, ruthenium (III) hexa mine, andquinone derivatives. Examples of suitable enzymes include glucoseoxidase, glucose dehydrogenase (GDH) based on pyrroloquinoline quinone(PQQ) co-factor, GDH based on nicotinamide adenine dinucleotideco-factor, and flavine-adenine dinucleotide (FAD) based GDH (FAD-GDH).One exemplary reagent formulation, which would be suitable for makingreagent layer 72, is described in pending U.S. application Ser. No.10/242,951, entitled, Method of Manufacturing a Sterilized andCalibrated Biosensor-Based Medical Device, published as U.S. PublishedPatent Application No. 2004/0120848, which is hereby incorporated byreference in its entirety.

Distal to the proximal sample chamber 61, body 59 can include connectiontracks that electrically connect first and second electrodes 166, 164with distal electrical contacts 63, 67. In one aspect, firstelectrically conductive layer 66 includes a first connection track 76that electrically connects first electrode 166 with a first electricalcontact 67. Similarly, second electrically conductive layer 64 caninclude a second connection track 78 that connects the second electrode164 with a second electrical contact 63 (FIG. 5A).

First and second electrically conductive layers can also define firstand second electrical contacts 67, 63 that facilitate electrical contactof test strip 62 with a test meter. In one embodiment, a portion offirst electrically conductive layer 66 extends distally from the distalend of spacer layer 60 and second electrically conductive layer 64 todefine first electrical contact 67. Second electrical contact can bedefined by a U-shaped notch 65 in the first electrically conductivelayer 66 which exposes a portion of second electrically conductive layer64. Applicants note that test strip 62 can include a variety ofalternative electrical contact configurations for electricallyconnecting to a test meter. For example, U.S. Pat. No. 6,379,513discloses electrochemical cell connection structures, and is herebyincorporated by reference in its entirety.

The sensors of FIGS. 4A through 5D can be used in conjunction with themeters, control units, and other components and steps of the devices,systems, and methods disclosed herein. Further disclosures related tothe electrochemical cells of FIGS. 4A through 5D are found in U.S.patent application Ser. No. 11/278,341 of Chatelier et al., entitled“Methods And Apparatus For Analyzing A Sample In The Presence OfInterferents,” and filed on March 31, the contents of which is herebyincorporated by reference in its entirety.

Another exemplary embodiment of a sample analyzing device for use inconjunction with at least some of the methods disclosed herein, animmunosensor 110, is illustrated in FIG. 6 and is described in U.S.patent application Ser. No. 12/570,268 of Chatelier et al., entitled“Adhesive Compositions for Use in an Immunosensor” and filed on Sep. 30,2009, the contents of which is hereby incorporated by reference in itsentirety. A plurality of chambers can be formed within the immunosensor,including a fill chamber, by which a sample can be introduced into theimmunosensor, a reaction chamber, by which a sample can be reacted withone or more desired materials, and a detection chamber, by which aconcentration of a particular component of the sample can be determined.These chambers can be formed in at least a portion of a lower electrode,an upper electrode, and a separator of the immunosensor. Theimmunosensor can also include a vent hole to allow air to enter andescape the immunosensor as desired, and first and second sealingcomponents to selectively seal first and second sides of the vent hole.The first sealing component can also form a wall of the fill chamber.

As illustrated, the immunosensor 110 includes a lower electrode 112having two liquid reagents 130, 132 striped onto it. The lower electrode112 can be formed using any number of techniques used to formelectrodes, but in one embodiment a polyethylene tetraphthalate (PET)sheet that is filled with barium sulphate is sputter-coated with gold.Other non-limiting example of forming an electrode are disclosed in U.S.Pat. No. 6,521,110 of Hodges et al., entitled “Electrochemical Cell” andfiled on Nov. 10, 2000, the contents of which is hereby incorporated byreference in its entirety.

Likewise, the liquid reagents 130, 132 can have a number of differentcompositions. In one embodiment the first liquid reagent 130 includes anantibody conjugated to an enzyme, such as GDH-PQQ, in a buffer thatcontains sucrose, as well as a poloxamer, such as Pluronics® blockcopolymers, an anticoagulant, such as citraconate, and calcium ions. Inone embodiment the second liquid reagent 132 includes a mixture offerricyanide, glucose, and a second mediator, such as phenazineethosulfate, in an acidic buffer, such as a dilute citraconic acidsolution. The first and second liquid reagents 130, 132 can be driedonto the lower electrode 112. A number of techniques can be used to drythe reagents 130, 132, but in one embodiment, following the striping ofthe reagents 130, 132 on the lower electrode 112, one or more infrareddryers can be applied to the reagents 130, 132. One or more air dryerscan also be used, for example, subsequent to the infrared dryers.References to a first reagent and a first liquid reagent and a secondreagent and a second liquid reagent herein are used interchangeably andare not necessarily an indication that the reagents are in their liquidor dried form at a given time for a particular embodiment. Further, someof the components associated with the first and second liquid reagentscan be used interchangeably and/or in both the first and second liquidreagents as desired. By way of non-limiting example, an anticoagulantcan be associated with either or both of the first liquid reagent 130and the second liquid reagent 132.

A line can be formed in the sputter-coated gold between the reagents130, 132 such that an edge of reagent 132 is very close to, or touches,the line. The line can be applied using laser ablation or with a sharpmetal edge. In one exemplary embodiment the line can be applied beforethe reagents 130, 132 are striped on the electrode. The line can bedesigned to electrically insulate the section of the lower electrode 112under the detection chamber from the section that will be under thereaction chamber. This can provide a better definition of an area of theworking electrode during the electrochemical assay.

The immunosensor 110 can also include an upper electrode 114 having oneor more magnetic beads 134 containing surface-bound antigens thereon.The antigens can be configured to react with the antibody disposed onthe lower electrode 112 and the sample within a reaction chamber 118, asdescribed in further detail below. Applicants note that the componentsdisposed on the lower electrode 112 and on the upper electrode 114 canbe interchangeable. Thus, the lower electrode 112 can include one ormore magnetic beads 134 and the upper electrode 114 can include twoliquid reagents 130, 132 striped onto it. Further, although in theillustrated embodiment the length of the electrode 112 forms the lengthof the entire body of the immunosensor 110, in other embodiments theelectrode can be only a portion of a layer of an immunosensor thatserves as the lower or upper electrode or multiple electrodes can bedisposed on a single layer of an immunosensor. Further, because voltageapplied to the immunosensor can be flipped and/or alternated, each ofthe lower and upper electrodes can serve as the working electrode andthe counter or counter/reference electrode at different stages. For easeof description purposes, in the present application the lower electrodeis considered the working electrode and the upper electrode the counteror counter/reference electrode.

A separator 116 disposed between the lower and upper electrodes 112, 114can have a variety of shapes and sizes, but it generally is configuredto desirably engage the lower and upper electrodes 112, 114 to form theimmunosensor 110. In one exemplary embodiment, the separator 116includes adhesive on both sides. The separator 116 can further include arelease liner on each side of the two sides of the separator 116. Theseparator 116 can be cut in a manner that forms at least two cavities. Afirst cavity can be formed to serve as a reaction chamber 118 and asecond cavity can be formed to serve as a detection chamber 120. In oneembodiment, the separator 116 can be kiss-cut such that the reactionchamber 118 is aligned with the electrodes 112, 114 to allow anantigen-antibody reaction therein while the detection chamber 120 isaligned with the electrodes 112, 114 to allow for the electrochemicaldetermination of ferrocyanide therein.

In one embodiment, the separator 116 can be placed on the lowerelectrode 112 in a manner that allows the magnetic beads 134 of theupper electrode 114 and the first reagent 130 of the lower electrode 112to be at least partially disposed in the reaction chamber 118 and theferricyanide-glucose combination of the second reagent 132 of the lowerelectrode 112 to be at least partially disposed in the detection chamber120. It can be advantageous to include an anticoagulant in each of thefirst and second liquid reagents 130, 132 so that an anticoagulant isassociated with each of the reaction and detection chambers 118, 120. Insome embodiments the combination of one of the upper and lowerelectrodes 112, 114 and the separator 116 can be laminated together toform a bi-laminate, while in other embodiments the combination of eachof the lower electrode 112, the upper electrode 114, and the separator116 can be laminated together to form a tri-laminate. Alternatively,additional layers may also be added.

A fill chamber 122 can be formed by punching a hole into one of thelower and upper electrodes 112, 114 and the separator 116. In theillustrated embodiment the fill chamber is formed by punching a hole inthe lower electrode 112 and the separator 116 such that the hole in thelower electrode 112 overlaps the reaction chamber 118. As shown, thefill chamber 122 can be a distance apart from the detection chamber 120.Such a configuration allows a sample to enter the immunosensor 110through the fill chamber 122 and flow into the reaction chamber 118 tobe reacted, for example with the first liquid reagent 130 that includesthe antibody conjugated to an enzyme in a buffer on the first electrode112 and the magnetic beads 134 striped on the upper electrode 114,without entering the detection chamber 120. Once the sample has beenreacted, it can then flow into the detection chamber 120 for interactionwith the second liquid reagent 132, for example the mixture offerricyanide, glucose, and the second mediator in an acidic buffer.

A vent 124 can be formed by punching a hole through each of the twoelectrodes 112, 114 and the separator 116 such that the vent 124 extendsthrough the entirety of the immunosensor 110. The hole can be formed ina suitable manner, such as, for example, drilled or punched in a numberof different locations, but in one exemplary embodiment it can overlap aregion of the detection chamber 120 that is spaced apart from thereaction chamber 118.

The vent 124 can be sealed in a number of different manners. In theillustrated embodiment, a first sealing component 140 is located on thelower electrode 112 to seal a first side of the vent 124 and a secondsealing component 142 is located on the upper electrode 114 to seal asecond side of the vent 124. The sealing components can be made ofand/or include any number of materials. By way of non-limiting example,either or both of the sealing components can be hydrophilic adhesivetape or Scotch® tape. Adhesive sides of the sealing components can facethe immunosensor 110. As shown, not only can the first sealing component140 form a seal for the vent 124, but it can also form a wall for thefill chamber 122 so that the sample can be contained therein. Propertiesincorporated onto the adhesive side of the first sealing component 140can be associated with the fill chamber 122. For example, if the firstsealing component 140 includes properties making it hydrophilic and/orwater soluble, the fill chamber can remain well-wet when a sample isdisposed therein. Further, the sealing components 140, 142 can beselectively associated and disassociated with the immunosensor 110 toprovide venting and/or sealing for the immunosensor 110 and thecomponents disposed therein as desired.

Adhesives can generally be used in the construction of the immunosensor.Non-limiting examples of ways in which adhesives can be incorporatedinto immunosensors and other sample analyzing devices of the presentdisclosure can be found in U.S. patent application Ser. No. 12/570,268of Chatelier et al., entitled “Adhesive Compositions for Use in anImmunosensor” and filed on Sep. 30, 2009, the contents of which wasalready incorporated by reference in its entirety.

While the present disclosure discusses a variety of differentembodiments related to immunosensors, other embodiments of immunosensorscan also be used with the methods of the present disclosure.Non-limiting examples of such embodiments include those described inU.S. Patent Application Publication No. 2003/0180814 of Hodges et al.,entitled “Direct Immunosensor Assay” and filed on Mar. 21, 2002, U.S.Patent Application Publication No. 2004/0203137 of Hodges et al.,entitled “Immunosensor” and filed on Apr. 22, 2004, U.S. PatentApplication Publication No. 2006/0134713 of Rylatt et al., entitled“Biosensor Apparatus and Methods of Use” and filed on Nov. 21, 2005, andU.S. patent application Ser. No. 12/563,091, which claims priority toeach of U.S. Patent Application Publication Nos. 2003/0180814 and2004/0203137, each of which is hereby incorporated by reference in itsentirety.

In one embodiment, the immunosensor 110 can be configured to be placedinto a meter that is configured to apply a potential to the electrodes112, 114 and measure a current that results from the application of thepotential. In one embodiment, the immunosensor includes one or more tabs117 for engaging a meter. Other features can also be used to engage theimmunosensor 110 with a meter. The meter can include a number ofdifferent features. For example, the meter can include a magnet that isconfigured to maintain certain components of the immunosensor 110 in onechamber while other components flow to the other. In one exemplaryembodiment, the magnet of the meter is located such that, upon placingthe immunosensor 110 in the meter, the magnet is disposed below thereaction chamber 118. This can allow the magnet to assist in holdingback any magnetic beads 134, and more particularly any antibody-enzymeconjugate that is bound to the beads 134, from flowing into thedetection chamber 120.

An alternate feature of the meter includes a heating element. A heatingelement can help speed up the reaction rate and help the sample flowthrough the immunosensor 110 in a desired manner by reducing theviscosity. A heating element can also allow one or more chambers and/ora sample disposed therein to be heated to a predetermined temperature.Heating to a predetermined temperature can help provide accuracy, forexample, by diminishing or removing the effects of temperature change asreactions occur.

Further, a piercing instrument can also be associated with the meter.The piercing instrument can be configured to pierce at least one of thefirst and second sealing components at a desired time so that air canflow out of the vent hole and liquid can flow from the reaction chamberinto the detection chamber.

The immunosensor 110 can also be configured to be associated with acontrol unit. The control unit can be configured to perform a variety offunctions. In one exemplary embodiment, the control unit is capable ofmeasuring a fill time of a sample when it is introduced to the device.In another embodiment, the control unit can be configured to determine ahaematocrit value of a blood sample. In yet another embodiment, thecontrol unit is configured to calculate a concentration of an analyte inthe sample in view of the fill time. In fact, the control unit caninclude a number of different features, depending, at least in part, onthe functionality desired and the method by which the system is designedto measure the fill time.

The control unit can also measure other aspects of the system. By way ofnon-limiting example, the control unit can be configured to measure atemperature of one or more chambers of the immunosensor. It can also beconfigured to measure a temperature of the sample, a color of thesample, or a variety of other characteristics and/or properties of thesample and/or the system. By way of further non-limiting example, thecontrol unit can be configured to communicate the results of the filltime determination, the results of the analyte concentrationdetermination, and/or the haematocrit measurement to outside equipment.This can be accomplished in any number of ways. In one embodiment, thecontrol unit can be hardwired to a microprocessor and/or a displaydevice. In another embodiment, the control unit can be configured towirelessly transmit data from the control unit to a microprocessorand/or a display device.

Other components of the system can also be configured to make suchmeasurements. For example, the immunosensor or the meter can beconfigured to measure a temperature of one or more chambers of theimmunosensor, measure or infer the temperature of a sample, or measure,determine, or infer a variety of other characteristics and/or propertiesof the sample and/or the system. Still further, Applicants note thatthese features of a control unit can be interchanged and selectivelycombined in a single control unit. For example, a control unit can bothdetermine a fill time and measure a temperature of a chamber. In otherembodiments, multiple control units can be used together to performvarious functions, based at least in part on the configurations of thevarious control units and the desired functions to be performed.

Example 1

The use of an electrochemical system to measure fill time isdemonstrated by the following example. In the following example, thesystem included a sensor with two opposed electrodes, with reagentsdesigned to react with the sample dried on one electrode. A plurality ofsamples was provided for analysis to test the performance of thesystems, devices, and methods disclosed herein. The samples were bloodsamples that contained three different levels of haematocrit, which wereknown so comparisons of the test results could be compared to the actualresults to determine the accuracy of the systems, devices, and methods.The four levels of haematocrit were approximately 20%, 60%, and 75%.Testing three levels of haematocrit allowed the accuracy of thedisclosed systems, devices, and methods to be confirmed over a broadspectrum of concentration levels.

In this example the electrode covered with the dried reagent is thesecond electrode. The first and second electrodes cover the entire areaof the chamber to be filled with liquid sample. Samples were introducedinto the sensor. While the introduction of samples into the sensor couldhave been accomplished in a variety of manners, in this example eachsample was admitted individually by way of capillary action into thefill chamber. As soon as the blood started to enter the detectionchamber, a 300 mV potential was applied to the electrodes by way of themeter for approximately four seconds. Alternatively, the voltage couldhave been applied prior to or while the blood was arriving in thedetection chamber. A plot of the current versus time transient resultingfrom this example is illustrated in FIG. 7. As shown in the FIG. 7, theline showing the time-current transient obtained with 75% haematocritblood is relatively flat from about 0.1 to about 0.5 seconds since thefilling process increases the area of the first electrode (which wouldtend to increase the current) and at the same time there iselectrochemical depletion of electroactive species at the firstelectrode (which would tend to decrease the current). These twoprocesses are approximately matched while the sensor is filling withblood. After fill is complete (at approximately 0.5 s) the first processis over and the second process dominates so that the current dropsabruptly. The latest time at which the current decreases sharply istaken as the fill time. The results for 20% and 60% haematocrit bloodshowed similar results, with a current drop at approximately 0.3 s for60% haematocrit blood and at approximately 0.1 s for 20% haematocritblood. The results of this experiment demonstrated the feasibility ofusing a measurement of current to determine the haematocrit percentageof blood.

Example 2

A second type of sensor was constructed which included two opposedelectrodes with reagents designed to react with the sample dried on oneelectrode. In this example however the electrode with the dried reagentwas the first electrode and was configured such that it did not coverthe entire area of the liquid filled chamber whereas the secondelectrode was configured such that it covered a wider area of the liquidfilled chamber and was contacted with liquid prior to the firstelectrode being contacted with liquid. When this sensor was used to testa plurality of blood samples adjusted to various haematocrits, thepattern of currents obtained shown in FIG. 8 was obtained. In thisExample, the four levels of haematocrit were approximately 30%, 44%, and62%. As shown in FIG. 8, the early part of each trace corresponds to theperiod during which the filling process increases the area of theworking electrode and hence increases the current. When the fill processis complete, the electrochemical depletion of electroactive speciestends to decrease the current at the time indicated by the arrows in thefigure. Once again, the time at which the current decreases sharply istaken as the fill time. The different configuration of the sensors leadsto a different dependence of fill time on haematocrit.

Example 3

The use of variable prepulse times in an electrochemical system isdemonstrated by the following example. A potentiostat meter wasconstructed which was capable of using the fill time information to varythe prepulse time using the methods discussed above. An initial test ofthe new meters was performed using heparinised capillary blood. Thenatural haematocrit and glucose were tested, and then plasma and 77%blood were tested at the natural or spiked glucose levels. Strips weretested on the original (fixed time) meters and on the meters whichincorporated the variable prepulse time algorithm disclosed above. Thedata were analyzed using the algorithm discussed above.

FIG. 9 shows that the 77% haematocrit blood gave negative biases (−19 to−28%) when tested with the original (fixed time) meters, but that allpoints were within 15% of the reference glucose measurement when testedwith the variable prepulse time meters. An example of a commerciallyavailable instrument configured to perform a reference glucosemeasurement is a Yellow Springs Instrument (YSI) glucose analyzer. Theoverall statistics for the two types of meters are summarized in Table1, below.

TABLE 1 Parameter Fixed time meters Variable time meters Mean CV (%) 3.63.0 Mean bias −9.4 −4.4 Global SD bias 12.0 5.9 % biases within 15% 62100

As shown in Table 1, the variable time meters outperformed the fixedtime meters in terms of accuracy and precision.

Example 4

The use of an electrochemical system to determine haematocrit on thebasis of fill time is demonstrated by the following example. In thisexample, the system included a sample analyzing device, in particularthe immunosensor 110 of FIG. 6, a meter configured to apply a potential,and a control unit configured to determine the initial fill velocity. Inparticular, a potential was applied to the electrodes of theimmunosensor 110, a level of haematocrit was determined, and then thepotential was reversed. The concentration of the analyte wassubsequently determined in view of the determined level of haematocrit.The level of haematocrit was determined in view of a fill time of thesample.

A plurality of samples was provided for analysis to test the performanceof the systems, devices, and methods disclosed herein. The samples wereblood samples that contained C-reactive proteins, and thus theconcentration of the analyte being determined was the concentration ofC-reactive proteins. The samples contained four different levels ofhaematocrit, which were known so comparisons of the test results couldbe compared to the actual results to determine the accuracy of thesystems, devices, and methods. The four levels of haematocrit wereapproximately 15%, 49%, 60%, and 72%. Testing four levels of haematocritallowed the accuracy of the disclosed systems, devices, and methods tobe confirmed over a broad spectrum of concentration levels.

In this example, an immunosensor was preheated to approximately 37° C.before a sample was introduced. The meter associated with theimmunosensor was configured to perform the preheating, although otheralternatives could have been used. Samples were then introduced into theimmunosensor. While the introduction of samples into the immunosensorcould have been accomplished in a variety of manners, in the exampleeach sample was admitted individually by way of capillary action intothe fill chamber.

After approximately two minutes had elapsed, the vent of theimmunosensor was accessed by piercing the first sealing component. Apiercing instrument of the meter was used to perform the piercingaction, which in turn allowed the blood to flow from the reactionchamber of the immunosensor into the detection chamber of theimmunosensor. As the blood entered the detection chamber, a 300 mVpotential was applied to the electrodes by way of the meter. As in theexamples discussed above, the current versus time transient was used todetermine the fill time of the sample according to the methods discussedabove. A plot of the fill time versus haematocrit percentage from thisexample is illustrated in FIG. 10. In some embodiments, the estimate ofthe haematocrit according to the methods disclosed herein can be used toexpress the antigen concentration with respect to plasma rather thanwhole blood, since this is more acceptable in pathology.

As discussed above, in some embodiments it may be desirable to onlymeasure a level of haematocrit. Thus, the first calculation based on theinitial current may be the only step that is needed to make thatcalculation. The actual determination of the haematocrit level can bedetermined as quickly as the initial current can be calculated. Thus, byway of non-limiting example, if the initial current is calculated basedon an average over the first 50 milliseconds, the level of haematocritcan be determined following the first 50 milliseconds. Thus,measurements of a haematocrit level of a blood sample can be performedin less than one second.

Example 5

An exemplary algorithm for correcting an analyte measurement based onfill time of a sample without further derivation of and correction forhematocrit is demonstrated by the following example. In this example, asensor which contained the enzyme FAD-GDH, instead of GDH-PQQ, wastested. A blood sample containing glucose was applied to the sensor andthe potential waveform shown in FIG. 11 was applied. A fill time of thesample was determined during the application of the first potential tothe sensor (E1, which was about +20 mV in this example) for about 1second. In this example, the fill time was determined to be the periodof time from the first detection of sample in the sensor until the timeat which the maximum value of the rate of change of the currenttransient during application of the first potential was measured, i.e.,the maximum value of i(t)−i(t+dt). The maximum value of i(t)−i(t+dt),i.e., the sharpest drop in current, corresponds to the time at which asufficient volume of the sample has filled the sensor for the analytemeasurement to be conducted. The fill time was not assessed during theapproximately first 0.15 seconds following sample detection, since theinitial signal is a combination of the rapid current decrease due to theconsumption of antioxidant species near the anode and the slower currentincrease which accompanies filling of the sensor. When these two ratesare matched then a pseudo steady state current is achieved and there islittle change in current while the rest of the sensor fills with blood.For this reason, the earliest fill time shown in FIG. 11 is about 0.15seconds.

Following application of the first potential (E1, for about 1 second), asecond test potential E2 of +300 mV was applied for about 3 secondsafter which a third test potential E3 of −300 mV was applied. Values ofi_(l) and i_(r) were calculated using Eqs. 2b and 3b. A value of i_(l)was calculated as the sum of currents from 3.9 to 4 seconds of the 5second long period and a value of i_(r) was calculated as the sum ofcurrents from 4.25 to 5 seconds of the 5 second long period. A firstglucose concentration in the sample was then calculated using Eq. 1,above. In this example, the values of p, a and zgr were 0.5796, 0.02722and 1.8, respectively.

The first glucose concentration was then corrected in view of the filltime of the sample according to Eqs. 14A, 14B, 15A, 15B, and 15C, above,for which the two threshold values of FT, Th₁ and Th₂ were 0.2 secondsand 0.4 seconds, respectively. As will be discussed in the followingexamples, Applicants found that the results of glucose measurementscorrected in view of fill time according to Eqs. 14A, 14B, 15A, 15B, and15C improved accuracy resulting in a lower bias from reference data.

Example 6

The dependence of bias from reference values of concentration on thefill time of samples is demonstrated in this example. Samples with arange of hematocrit from about 0 to about 70% were tested using FAD-GDHsensors according to the algorithms discussed above, but were notcorrected for fill time. FIG. 12 shows that the bias of samples fromreference values of analyte concentration was dependent on the fill timeof the sample. For example, as shown in FIG. 12, the bias of samples wasincreasingly negative as fill time increases. In other words, theaccuracy of uncorrected values of analyte concentration decreased forsamples with longer fill times. Thus, there is a distinct dependence ofthe bias on the fill time of samples.

Example 7

The improvement resulting from correcting analyte concentration in viewof fill time is demonstrated in this example. FIG. 13A shows the samedata set as shown in FIG. 12 plotted against the hematocrit range of thesamples. FIG. 13B shows the improvement obtained when the data iscorrected in view of fill time according to Eqs. 14A, 14B, 15A, 15B, and15C, above. As illustrated in FIGS. 13A and 13B, the global SD biasdecreased from 6.2 to 5.7 after the data was corrected for fill time.Thus, correcting for fill time according to the above algorithmsprovides improved accuracy.

Example 8

Increased accuracy using fill time correction in a clinical setting isdemonstrated by this example. FIG. 14 illustrates a plot of the biasversus fill time data for samples obtained from 311 donors tested usingFAD-GDH sensors in a clinical setting according to the algorithmsdiscussed above in Example 5. For this data set, the fill timecorrection provided a decrease in global SD bias from 5.75 to 5.58. Theimprovement in this clinical data was only modest because most samplesfilled the sensor in about 0.2 seconds or less, and were thisuncorrected by the fill time algorithm.

Example 9

The data in the previous examples were obtained at 50 ms data density(i.e., one current value was stored every 50 ms). Better resolution infill times can be obtained with faster data storage, e.g., 10 ms datadensity, as shown in FIG. 15. FIG. 15 illustrates the current transientsobtained when blood with hematocrits in the range of about 15% to about72% was loaded into sensors. FIG. 16 illustrates fill time datacalculated from the data of FIG. 15. FIG. 16 shows the raw fill timevalues as open diamonds, the mean of 5 replicates as filled squares, and±1 SD as vertical bars. As shown in FIG. 16, the fill times ranged fromabout 0.06 seconds to about 0.32 seconds, with higher hematocrit samplesfilling more slowly. When the data presented in this example was testedfor glucose concentration, the global SD bias decreased from 5.08 to4.71 after the glucose values were corrected for fill time using thealgorithms discussed above in Example 5.

Applicants note that these nine examples are merely nine of manyexamples of how the teachings contained herein can be performed andused. Further, although the methods, systems, and devices disclosedherein are primarily used in conjunction with determining aconcentration of an analyte of a blood sample, and are primarily focusedon accounting for errors that can result from varying fill times andlevels of haematocrit in blood samples, Applicants note that thedisclosures contained herein can also be used for a variety of othersamples containing analytes and can test for a variety of antigensand/or antibodies contained within a sample.

Applicants note that to the extent various methods, systems, and devicesrely on a particular equation, the equations provided are generallybased on the examples to which the equations were applied. One skilledin the art, in view of the present disclosure, will be able to makeadjustments to the disclosed equations for other situations withoutdeparting from the scope of the invention.

Still further, the methods discussed herein, such as those related todetermining a concentration and using the systems and devices, are alsonot limited by the particular steps or order of the steps, except whereindicated. One skilled in the art will recognize various orders in whichthe methods can be performed, and further, will recognize that steps canbe modified or added without departing from the scope of the invention.

Non-limiting examples of some of the other types of devices with whichthe methods disclosed herein can be used are discussed in greater detailin U.S. Pat. No. 5,942,102 of Hodges et al., entitled “ElectrochemicalMethod” and filed on May 7, 1997, U.S. Pat. No. 6,174,420 of Hodges etal., entitled “Electrochemical Cell” and filed on May 18, 1999, U.S.Pat. No. 6,379,513 of Chambers et al., entitled “Sensor ConnectionMeans” and filed on Sep. 20, 1999, U.S. Pat. No. 6,475,360 of Hodges etal., entitled “Heated Electrochemical Cell” and filed on Sep. 11, 2000,U.S. Pat. No. 6,632,349 of Hodges et al, entitled “Hemoglobin Sensor”and filed on Jul. 14, 2000, U.S. Pat. No. 6,638,415 of Hodges et al.,entitled “Antioxidant Sensor” and filed on Jul. 14, 2000, U.S. Pat. No.6,946,067 of Hodges et al., entitled “Method of Forming an ElectricalConnection Between an Electrochemical Cell and a Meter” and filed onDec. 9, 2002, U.S. Pat. No. 7,043,821 of Hodges, entitled “Method ofPreventing Short Sampling of a Capillary or Wicking Fill Device” andfiled on Apr. 3, 2003, and U.S. Pat. No. 7,431,820 of Hodges et al.,entitled “Electrochemical Cell” and filed on Oct. 1, 2002, each of whichis hereby incorporated by reference in its entirety.

Further, to the extent the disclosures herein are discussed for use witha device having a particular configuration, any number of configurationscan be used. For example, some configurations that can be used with thepresent disclosures include sensors having two electrodes facing eachother, sensors having two electrodes on the same plane, and sensorshaving three electrodes, two of which are opposed and two of which areon the same plane. These different configurations can occur in anynumber of devices, including immunosensors and the other aforementioneddevices.

Various aspects of the devices, systems, and methods can be adapted andchanged as desired for various determinations without departing from thescope of the present invention. Further, one skilled in the art willappreciate further features and advantages of the invention based on theabove-described embodiments. Accordingly, the invention is not to belimited by what has been particularly shown and described, except asindicated by the appended claims. All publications and references citedherein are expressly incorporated herein by reference in their entirety.

1. A method for determining a concentration of an analyte in a sample,the method comprising: detecting a presence of the sample in anelectrochemical sensor, the electrochemical sensor comprising twoelectrodes; determining a fill time of the sample with the twoelectrodes; calculating a correction factor in view of at least the filltime; reacting an analyte to cause a physical transformation of theanalyte between the two electrodes; and determining the concentration ofthe analyte in view of the correction factor with the same twoelectrodes.
 2. The method of claim 1, wherein determining the fill timeof the sample comprises: applying an electric potential between the twoelectrodes while the sample is introduced; measuring a current as afunction of time; and determining a current drop time based on thecurrent as a function of time, wherein the current drop time correspondsto the fill time of the sample.
 3. The method of claim 2, whereindetermining the current drop time comprises calculating the maximumnegative value of the change in the measured current over time.
 4. Themethod of claim 2, wherein determining the current drop time comprisescalculating a difference between at least two current values where thedifference is greater than a first predetermined threshold.
 5. Themethod of claim 2, wherein determining the current drop time comprisescalculating a difference between at least two current values where thedifference is less than a second predetermined threshold.
 6. The methodof claim 2, wherein determining the current drop time comprisescalculating a slope in the measured current as a function of time wherethe slope is greater than a third predetermined threshold.
 7. The methodof claim 2, wherein determining the current drop time comprisescalculating a slope in the measured current as a function of time wherethe slope is less than a fourth predetermined threshold.
 8. The methodof claim 2, wherein determining the current drop time comprisescalculating an inflection point in the measured current as a function oftime.
 9. The method of claim 1, in which the detecting the presence ofthe sample comprises: applying an electric potential between the twoelectrodes, and measuring a change in current values that is greaterthan a fifth predetermined threshold.
 10. The method of claim 1, inwhich the detecting the presence of the sample comprises: applying anelectric potential between the two electrodes, and measuring a change incurrent values that is less than a sixth predetermined threshold. 11.The method of claim 1, in which the detecting the presence of the samplecomprises: applying a generally constant current between the twoelectrodes, and measuring a change in an electric potential that isgreater than a seventh predetermined threshold.
 12. The method of claim1, in which detecting the presence of the sample comprises: applying agenerally constant current between the two electrodes, and measuring achange in an electric potential that is less than an eighthpredetermined threshold.
 13. The method of claim 1, in which detectingthe presence of the sample is performed by a microprocessor of ananalyte measuring machine.
 14. The method of claim 1, in which reactingof the analyte generates an electroactive species that is measured as acurrent by the two electrodes.
 15. The method of claim 1, in which thetwo electrodes comprise an opposing faced orientation.
 16. The method ofclaim 1, in which the two electrodes comprise a facing orientation. 17.The method of claim 1, wherein the electrochemical sensor comprises aglucose sensor
 18. The method of claim 1, wherein the electrochemicalsensor comprises an immunosensor.
 19. The method of claim 1, wherein thesample comprises blood.
 20. The method of claim 1, wherein the samplecomprises whole blood.
 21. A method for measuring a corrected analyteconcentration, the method comprising: detecting a presence of the samplein an electrochemical sensor, the electrochemical sensor comprising twoelectrodes; determining a fill time of the sample with the twoelectrodes; reacting an analyte to cause a physical transformation ofthe analyte; determining a first analyte concentration in the samplewith the same two electrodes; and calculating a corrected analyteconcentration based on the first analyte concentration and the filltime.
 22. The method of claim 21, wherein the step of calculating thecorrected analyte concentration comprises: calculating a correctionfactor based on the fill time, wherein the corrected analyteconcentration is calculated based on the first analyte concentration andthe correction factor.
 23. The method of claim 22, wherein thecorrection factor comprises about zero when the fill time is less than afirst fill time threshold.
 24. The method of claim 22, wherein thecorrection factor is calculated in view of the fill time when the filltime is greater than a first fill time threshold and less than a secondfill time threshold.
 25. The method of claim 22, wherein the correctionfactor comprises a constant value when the fill time is greater than asecond fill time threshold.
 26. The method of claim 22, wherein the stepof calculating the corrected analyte concentration comprises calculatinga sum of the correction factor and the first analyte concentration inthe sample when the first analyte concentration in the sample is lessthan a threshold value.
 27. The method of claim 22, wherein the step ofcalculating the corrected analyte concentration when the first analyteconcentration in the sample is greater than a threshold value comprises:dividing the correction factor by one hundred and adding one to give anintermediate term; and multiplying the intermediate term by the firstanalyte concentration to give a fill time corrected analyteconcentration.
 28. The method of claim 21, wherein determining the filltime of the sample comprises: applying an electric potential between thetwo electrodes while the sample is introduced; measuring a current as afunction of time; and determining a current drop time based on thecurrent as a function of time, wherein the current drop time correspondsto the fill time of the sample.
 29. The method of claim 28, whereindetermining the current drop time comprises calculating the maximumnegative value of the change in the measured current over time.
 30. Themethod of claim 28, wherein determining the current drop time comprisescalculating a difference between at least two current values where thedifference is greater than a first predetermined threshold.
 31. Themethod of claim 28, wherein determining the current drop time comprisescalculating a difference between at least two current values where thedifference is less than a second predetermined threshold.
 32. The methodof claim 28, wherein determining the current drop time comprisescalculating a slope in the measured current as a function of time wherethe slope is greater than a third predetermined threshold.
 33. Themethod of claim 28, wherein determining the current drop time comprisescalculating a slope in the measured current as a function of time wherethe slope is less than a fourth predetermined threshold.
 34. The methodof claim 28, wherein determining the current drop time comprisescalculating an inflection point in the measured current as a function oftime.
 35. The method of claim 21, in which detecting the presence of thesample comprises: applying an electric potential between the twoelectrodes, and measuring a change in current values that is greaterthan a fifth predetermined threshold.
 36. The method of claim 21, inwhich detecting the presence of the sample comprises: applying anelectric potential between the two electrodes, and measuring a change incurrent values that is less than a sixth predetermined threshold. 37.The method of claim 21, in which detecting the presence of the samplecomprises: applying a generally constant current between the twoelectrodes, and measuring a change in an electric potential that isgreater than a seventh predetermined threshold.
 38. The method of claim21, in which detecting the presence of the sample comprises: applying agenerally constant current between the two electrodes, and measuring achange in an electric potential that is less than an eighthpredetermined threshold.
 39. The method of claim 21, in which detectingthe presence of the sample is performed by a microprocessor of ananalyte measuring machine.
 40. The method of claim 21, in which reactingof the analyte generates an electroactive species that is measured as acurrent by the two electrodes.
 41. The method of claim 21, in which thetwo electrodes comprise an opposing faced orientation.
 42. The method ofclaim 21, in which the two electrodes comprise a facing orientation. 43.An electrochemical system, comprising: (a) an electrochemical sensorincluding electrical contacts configured to mate with a test meter, theelectrochemical sensor comprising: (i) a first electrode and a secondelectrode in a spaced apart relationship, and (ii) a reagent; and (b)the test meter including a processor configured to receive current datafrom the test strip upon application of voltages to the test strip, andfurther configured to determine a corrected analyte concentration basedon a calculated analyte concentration and a measured fill time with thesame two electrodes.
 44. The electrochemical system of claim 43, whereinthe test meter includes data storage containing an analyte concentrationthreshold, a first fill time threshold, and a second fill timethreshold.
 45. The electrochemical system of claim 43, furthercomprising a heating element configured to heat at least a portion ofthe electrochemical sensor.
 46. The electrochemical system of claim 43,wherein the electrochemical sensor comprises a glucose sensor.
 47. Theelectrochemical system of claim 43, wherein the electrochemical sensorcomprises an immunosensor.
 48. The electrochemical system of claim 43,wherein at least one of the electrochemical sensor, the test meter, andthe processor are configured to measure a temperature of the sample. 49.The electrochemical system of claim 43, wherein the analyte comprisesC-reactive protein.
 50. The electrochemical system of claim 43, whereinthe analyte comprises glucose.
 51. The electrochemical system of claim43, wherein the sample comprises blood.
 52. The electrochemical systemof claim 43, wherein the sample comprises whole blood.
 53. Theelectrochemical system of claim 43, in which the first and secondelectrodes comprise an opposing faced orientation.
 54. Theelectrochemical system of claim 43, in which the first and secondelectrodes comprise a facing orientation.